Nanofiber structure with immobilized organic substance and the method of its preparation

ABSTRACT

The invention concerns a nanofiber structure with immobilized organic agens that consists of silica nanofibers whose surface was modified with aminoaikyialkoxysilane and of subsequently immobilized organic agens. The invention also concerns a method of preparation of the nanofiber structure with immobilized organic agens, while silica nanofibers are prepared by electrostatic spinning from the initial sol synthesized by a sol-gel method from tetraalkoxysilane, heat-treated and their the surface modified by a solution of aminoaikyialkoxysilane and organic agens is then immobilized on the modified surface of the nanofibers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §§371 national phase conversionof PCT/CZ2012/000128, filed Dec. 10, 2012, which claims priority toCzech Republic Patent Application No. PV 2012-549, filed Aug. 14, 2012,the contents of which are incorporated herein by reference. The PCTInternational Application was published in the English language.

TECHNOLOGY AREA

The invention concerns a nanofiber structure with immobilized organicsubstance.

The invention also concerns a method of preparation of the nanofiberstructure with immobilized organic substance.

EXISTING STATE OF TECHNOLOGY

In many medicine and biotechnology applications functional organicsubstance (medicines, enzymes etc.) is applied by being placed into therespective environment where they should operate, i.e. into wounds, orbiochemical reactors etc. Sometimes the functional organic substanceneed to be significantly overdosed because there are significant lossesof the substance due to dispersion or washing away (typically washing ofantibiotics from the wound etc.) or it is necessary to administer thesubstance frequently. This, however, may cause permeation of thesubstance into other parts of the organism and undesired side effectsor, on the contrary, insufficient efficiency of the applied substance.

In biochemical applications the employed organic substance (mostlyenzymes) usually cannot be recovered from the application area, e.g. forfurther use, and a new quantity of the functional organic substanceneeds to be added into the next production batch. This, however,significantly increases costs and the reaction product contains freeenzyme which is not desirable from the viewpoint of further product use.

These problems may be eliminated or limited by immobilization of organicsubstance on suitable substrates, i.e. ideally by permanent or at leastas long as possible lasting attachment of organic substance on suitablecarriers. The preconditions of this approach include, apart from asufficiently stable immobilization of the organic substance on thesubstrate, keeping of the efficiency and function of the substance andalso a sufficient quantity of the immobilized substance.

The quantity of the immobilized substance depends on the number ofsuitable bonding places for immobilization and on the specific surfaceof the substrate. From this point of view nanofibers are a particularlysuitable substrate because their specific surface is in units to tens ofm²/g while they keep suitable mechanical properties; those propertiesmake it possible to form shapeable nanofiber layers that may be easilyplaced into a wound or into a holder in a biochemical reactor and aftercompletion of the application they may be removed.

The patents CZ 294274 or WO 2005024101 describe a method of nanofiberpreparation by electrostatic spinning. However, the patents CZ 294274 orWO 2005024101, do not specify in detail polymer solutions forpreparation of the nanofibers.

In the patent WO2009018104 methyltrimethoxysilane is used as a precursorto prepare silica nanofibers. Without heat treatment according toWO2009018104 or if heat-treated at low temperatures according toWO2009018104, the nanofibers will have hydrophobic properties as aresult of presence of methyl groups on the surface and the number ofSi—OH groups on the surface for its subsequent potential modificationwith aminoalkylalkoxysilane will be low. For this reason the procedureunder WO 2009018104 is not suitable for preparation of initial silicananofibers for the subsequent stages of modification and immobilizationof organic substance.

The patents JP20040041335, JP20040161234 and JP20040243580 describepreparation of an organic-inorganic nanofiber composite made of aregular framework of polyethylene imide fibers with layers of silicondioxide applied with a sol-gel method. The resulting composite materialis intended to trap, or to increase concentrations of, varioussubstances in the prepared structure, however, only as a filter, i.e. ingaps between individual nanofibers, or by simple adsorption of thedesired particles in the bulk nanofiber composite.

The packaging paper under the patent KR20090058155 is made of nanofibersobtained by electrostatic spinning of a biodegradable organic polymerwith addition of sol of silicon dioxide and silver nitrate. Theresulting product has antiseptic and antibacterial properties but it isnot suitable for immobilization of organic substance.

The patent KR20100058372 describes preparation of a catalyst frommesoporous nanofibers of silicon dioxide prepared by growing from agaseous phase and subsequent introduction of a catalyst with silane onthe surface and into the pores of the nanofibers prepared in thismanner. The resulting product is described as a catalyst of variousorganic reactions and it is not used for immobilization of organicsubstance.

The drawback of the existing state of technology consists in the absenceof a sufficiently biochemically stable structure capable of sufficientlyhigh, efficient and, in terms of time, stable immobilization of organicsubstance.

The objective of this invention is to eliminate, or at least tominimize, disadvantages of the current state of technology.

PRINCIPLE OF THE INVENTION

The objective of the invention is achieved by a nanofiber structure withimmobilized organic substance, the principle of which consists in thefact that silica nanofibers have their surface modified by a reactionwith aminoalkylalkoxysilane and subsequently organic substance isimmobilized on their surface with peptide or hydrogen bonds.

Silica nanofibers are suitable particularly thanks to their highstability in biochemical reactions and thanks to their dissolvingability in body fluids. The dissolving rate of silica nanofibers iscontrolled by the temperature of thermal processing of the silicananofibers and therefore the nanofibers may be removed from the woundalong with the immobilized substance after a previously specified timeand the residues of silica nanofibers potentially released in the placeof application, in a wound or bioreactor, e.g. by breaking etc., arethen dissolved in body fluids or in the bioreactor sufficiently quicklywithout any negative side effects. Silica nanofibers have anotheradvantage that their surface with high numbers of Si—OH groups can beeasily modified with reactions in which Si—OH groups are linked viacovalent bonds with aminoalkylalkoxysilanes whose amino groupssubsequently enable formation of relatively strong peptide bonds orslightly weaker hydrogen bonds with the immobilized organic substance.

The principle of the method of preparation of the nanofiber structurewith immobilized organic substance consist in preparation of an initialsol from tetraalkoxysilane using a sol-gel method and the sol is thenexposed to electrostatic spinning; the resulting nanofiber structure isheat-treated and its surface is treated with aminoalkylalkoxysilane; thesurface of the nanofiber structure is then exposed to a solution organicsubstance and the organic substance is immobilized by means of peptideor hydrogen bonds on the surface of the nanofiber structure.

The basis of this method is to create a nanofiber structure, e.g. inform of silica nanofibers made by electrostatic spinning from a solprepared by a sol-gel method from tetraalkoxysilane using acidiccatalysis and without additional organic polymers. In order to obtain asol with suitable properties for electrostatic spinning it is necessaryto observe the molar ratio of water to tetraalkoxysilanek=[H₂O]/[tetraalkoxysilane] in the range k=1.6 to 3, the molar ratio ofacid to tetraalkoxysilane m=[HA]/[tetraalkoxysilane] in the rage m=0.001to 1 and the sol shall be concentrated before spinning by evaporation ofthe solvent to achieve the SiO₂ concentration in the sol in the range 28to 44 wt. %.

This method can be used to prepare silica nanofibers with the averagediameter in the range 100 to 1000 nm (depending on the conditions ofpreparation of the initial sol and the conditions of electrostaticspinning) to form a nanofiber structure (layer) that can be directlyused for subsequent operations, without the necessity to remove anyadditives by heat treatment at high temperatures. Specific surfaces ofnanofiber structures (layers) prepared in this manner range from unitsto tens of m²/g and thus they ensure large surfaces for immobilizationof organic substance even if the nanofiber layer is thin.

The resulting properties of the nanofiber structure, e.g. the number ofactive Si—OH groups for subsequent bonds with aminoalkylalkoxysilane andchemical durability against water and body fluids, are fundamentallyaffected by a sufficient heat treatment of the nanofiber layer beforeimmobilization of organic substance. The morphology of nanofiberspractically does not change up to the temperature around 850° C., whenit transforms into silica glass (except a slight reduction of theirdiameters as a result of their thickened structure at hightemperatures). During the heat treatment silica nanofibers graduallyreduce their chemical solubility and the number of active Si—OH groupsfor subsequent bonds with aminoalkylalkoxysilane. Sufficient chemicalsolubility of silica nanofibers in body fluids is necessary because thesize of nanofiber fragments released during manipulation with thenanofiber structure and their accidental inhalation is in the area ofdocumented carcinogenity in case of their long-term local presence (morethan 40 days). During dynamic and static tests of silica nanofibersdissolving (average diameter around 180 nm) in simulated lung fluid thedissolving rate of nanofibers was strongly dependent on the temperatureof thermal processing of the nanofibers. In case of heat treatment atlow temperatures (180° C./2 hours) the nanofibers dissolved within 7days and they can be considered safe for manipulation and in case ofaccidental inhalation. However, in case of heat treatment at highertemperatures the nanofibers gradually became less soluble and thenanofibers heat-treated in this manner shall be viewed as potentiallycarcinogenic.

Most of medically of biochemically active organic substance containcarboxyl (—COON) or at least hydroxyl (—COH) groups. However, to ensurea sufficient level and reliability of immobilization of such organicsubstance on the surface of silica nanofibers it is necessary to modifythe surface of the nanofibers so that there are firmly bound aminogroups on it to form peptide bonds with carboxyl groups or hydrogenbonds with hydroxyl groups. In this manner the organic substance issufficiently immobilized on the surface of nanofibers. The surface ofnanofibers is therefore modified by a solution of aminoalkylalkoxysilanewhich forms covalent bonds, by polycondensation via alkoxy groups, withsurface Si—OH groups of silica nanofibers and with its free aminoalkylgroup provides a primary functional amino group for the formation ofpeptide or hydrogen bonds with organic substance. In a suitable solventor environment (water, alcohol or other organic solution) the formationof bonds between amino groups and organic substance is spontaneous. Theimmobilization of organic substance on the surface of silica nanofiberswith the surface modified according to this invention is sufficientlystrong to ensure that during the subsequent application of the nanofiberstructure with immobilized organic substance, e.g. in presence of wateror body fluids, like in open wounds etc., the immobilized organicsubstance operates but it is not released from the nanofiber structureor is released only in small quantities, i.e. only in minimum quantitiesThis ensures a long-term, contact and high concentration of organicsubstance at a place of their desired application, without being washedaway and without excessive release from the place of application.

EXAMPLES OF EXECUTION OF THE INVENTION

The invention will be described on examples of the procedures to preparenanofiber structure in form of a layer of silica nanofibers withmodified surface and immobilized organic substance on the surface. Inthe following text the invention is documented with specific exampleswhich, however, do not document all possibilities of the invention whoseapplication and use are obvious to an average expert from this textwithout any additional inventing efforts.

Example 1

The initial sol for preparation of silica nanofibers was prepared with amodified sol-gel method. 400 ml of tetraethoxysilane were dissolved in330 ml isopropyl alcohol and water and HCl were added to achieve themolar ratio k=[H₂O]/[tetraalkoxysilane]=2.3 and the molar ratiom=[HCl]/[tetraalkoxysilane]=0.01. After completion of hydrolytic andpolycondensation reactions the sol was concentrated by evaporation ofthe solvent to 36 wt. % of SiO₂.

The prepared sol was used for electrostatic spinning at the voltage of50 kV and the distance of 15 cm. The average size of the preparednanofibers was 180 nm. The resulting nanofiber structure in from of alayer of nanofibers was heat-treated at 180° C. for 2 hours in a dryingkiln and the surface of silica nanofibers was modified by immersion intoa 2% solution of 3-aminopropyltriethoxysilane in anhydrous ethanol for 1hour at the laboratory temperature. The modified nanofiber structure waswashed three times with anhydrous ethanol and submerged into 2% solutionof tetracycline or penicillin in anhydrous ethanol for 2 hours at thelaboratory temperature. Finally, the nanofiber structure with theimmobilized antibiotic (organic substance) was flushed twice withanhydrous ethanol and left to dry in a desiccator.

The function of the nanofiber structure with the immobilized antibioticwas verified by antibacterial tests on a selected group of pathogenicbacterial strains that may cause problems particularly in dermatology.They included bacterial strains Staphylococcus aureus, MRSA, Escherichiacoli, Klebsiella pneumoniae, Proteus vulgaris, Proteus mirabilis andPseudomonas aeruginosa. Samples of the nanofiber structure with theimmobilized antibiotic were placed into a center of a Petri dish withrespective bacterial inoculum of a selected bacterial strain. Thesamples were incubated in a thermostat for 24 hours at 37° C. Further,the size of the so-called halo zones was evaluated (i.e. zones aroundthe nanofiber structure with immobilized antibiotics).

Results of the antibacterial tests were excellent—the sizes of testedhalo zones were evaluated as 100% inhibition ability of the antibioticsagainst the selected bacterial pathogenic strains. This has confirmedthat both the antibiotics were immobilized on the nanofiber structure ofsilica nanofibers with modified surface hereunder, without losing theirfunction.

Example 2

The initial sol for preparation of silica nanofibers was prepared by amodified sol-gel method. 400 ml of tetraethoxysilane were dissolved in330 ml isopropyl alcohol and water and HCl were added to achieve themolar ratio k=[H₂O]/[tetraalkoxysilane]=2.0 and the molar ratiom=[HCl]/[tetraalkoxysilane]=0.01. After completion of hydrolytic andpolycondensation reactions the sol was concentrated by evaporation ofthe solvent to 40 wt. % of SiO₂.

The prepared sol was used for electrostatic spinning at the voltage of50 kV and the distance of 15 cm. The average size of the preparednanofibers was 580 nm. The resulting nanofiber structure was in from ofa layer of nanofibers was heat-treated at 180° C. for 2 hours in adrying kiln and the surface of nanofibers was subsequently modified witha 2% solution of 3-aminopropyltrimethoxysilane in distilled water for 1hour at the laboratory temperature. The nanofiber structure with themodified surface was washed three times with distilled water and oncewith a solution of phosphate buffer (pH=7.2) and then it was submergedinto a 2% solution of esterase or lipase enzyme in phosphate buffer for10 minutes at the laboratory temperature. Finally, the nanofiber layerwith the immobilized enzyme was flushed two times with phosphate bufferand left to dry in the laboratory environment.

The immobilization of the enzymes on the nanofiber structure wasverified with a histochemical azocopulation reaction of alpha-naphtylacetate and chromogenic dye with the enzyme. The solution ofalpha-naphtyl acetate and Fast Blue BB dye in phosphate buffer incombination with the enzyme formed colored deposits of immobilizedenzyme that were visible in a microscope and demonstrated its presence.The tests were performed as comparative against a nanofiber structuremade of silica nanofibers on which no enzyme was immobilized hereunder,while no deposits as described above were found on the control nanofiberstructure without the enzymes.

1. A nanofibrous structure with immobilized organic substance composedof pure silica nanofibres made by electrostatic spinning, the nanofibershave controlled dissolution in body fluids or in bioreactor and havesurface modified by aminoalkylalkoxysilane with subsequently immobilizedorganic agent substances.
 2. The method of production of nanofibrousstructure with immobilized organic substance, wherein, from the initialsol synthesized by the sol-gel method from tetraalkoxysilane by means ofelectrostatic spinning the pure silica nanofibres are created, which aresubsequently heat treated for controlled dissolution in body fluids orin bioreactor and then surface of nanofibres is modified by means ofsolution of aminoalkylalkoxysilane, after which to such modified surfaceof nanofibres the organic substances are immobilized.
 3. The methodaccording to the claim 2, wherein the initial sol is prepared by thesol-gel method form solution of tetraalkoxysilane in alcohol withaddition of water upon an acid catalysis by controlled hydrolysis andpolycondensation and by thickening through distilling-off the solvent toa viscosity necessary for electrostatic spinning.
 4. The methodaccording to the claim 3, wherein the alcohol is ethanol orisopropylalcohol, an acid catalysis of hydrolysis and polycondensationof tetraalkoxysilane is ensured by addition of inorganic or organicacid, the initial molecular ratio of water to tetraalkoxysilanek=[H₂O]/[tetraalkoxysilane] is within values k=1.6 to 3, molecular ratioof an acid to tetraalkoxysilane m=[HA]/[tetraalkoxysilane] is withinvalues m=0.001 to 1 and the sol before spinning is thickened byevaporation of the solvent to the content of SiO₂ in the sol to bewithin values 28 to 44% by weight.
 5. The method according to the claim4, wherein the tetraalkoxysilane is tetraethoxysilane and the acid ishydrochloric acid or nitric acid.
 6. The method according to the claim2, wherein through the electrostatic spinning purely silica nanofibreshaving mean diameter 100 to 1000 nm are prepared, which are then heattreated within temperature 30 to 900° C. for a period of 10 minutes to10 hours.
 7. The method according to the claim 6, wherein the puresilica nanofibres are heat treated at a temperature within 150° C. to250° C. for a period 1 to 3 hours.
 8. The method according to the claim2, wherein the solution of aminoalkylalkoxysilane is in water, alcoholor other organic solvent and it has concentration of 0.1 to 10% byweight.
 9. The method according to the claim 8, wherein theaminoalkylalkoxysilane is 3-aminopropyltriethoxysilane or3-aminopropyltrimethoxysilane.
 10. The method according to the claim 2,wherein the organic agent substance is immobilized to the modifiedsurface of nanofibres in the liquid environment in a period of 30seconds to 48 hours.
 11. The method according to the claim 10, whereinthe liquid environment is water, aqueous solution of biochemical buffer,ethanol, isopropylalcohol or acetone and immobilization time varies from3 minutes to 24 hours.